Using non-precipitating monospecific antibodies we are studying the conformations of hemoglobins in solutions. Hemoglobin A and A2 have been isolated, purified and isotopically labeled either by carbamoylation with K14CNO or by reductive alkylation with tritiated borohydride. A specific subpopulation of anti-hemoglobin antibodies has been isolated by affinity chromatography using a synthetic peptide corresponding to alpha(129-141) and has been used in a radioimmunoassay to study conformational changes that occur during oxygenation at the carboxy-terminus of aplha chains. These antibodies bind with an affinity of 5 times 10 to the 8th power M-1 to CO-liganded (C) hemoglobin and increase the oxygen affinity of hemoglobin. A second subpopulation of antibodies has been isolated that react specifically with 3(H)-hemoglobin A2. These antibodies have been used in a radioimmunoassay to detect hemoglobin A2 levels in serum and cells and to differentiate A2 from other normal and mutant hemoglobins. In addition to these immunologic studies, we have undertaken investigation into the role of 2,3-DPG as a regulator of hemoglobin oxygen affinity. In our initial studies we have purified the three enzymes that regulate intracellular 2,3 DPG levels and are conducting further physico-chemical studies of them.